Mechanisms and regulation of macropinosome closure

大胞质体关闭的机制和调控

• 批准号: 7509465
• 负责人: JOEL A SWANSON
• 金额: $ 18.65万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-21 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7509465
• 关键词: 1-Phosphatidylinositol 4-Kinase; 3-Dimensional; Actins; Binding; Cell membrane; Cell surface; Cells; Chimera organism; Clathrin; Clathrin-Coated Vesicles; Coated vesicle; Cytoplasm; Cytoskeleton; Diffusion; Disease; Distal; Dynamin; Endocytic Vesicle; Epithelial Physiology; Event; Fluorescence; Fluorescence Microscopy; Fluorescent Probes; Guanosine Triphosphate Phosphohydrolases; Health; Host Defense; Immunity; Infection; Lateral; Life; Liquid substance; Maps; Measures; Mechanics; Membrane; Methods; Microscopic; Molecular; Morphology; Movement; Myosin ATPase; Organelles; Phagocytosis; Phagosomes; Phase; Phosphatidylinositols; Phospholipids; Phosphotransferases; Pinocytosis; Process; Quantitative Microscopy; Regulation; Renal function; Shapes; Staging; Structure; Tertiary Protein Structure; Testing; Time; Vesicle; Work; base; constriction; drinking; instrument; macrophage; novel; prevent; public health relevance; reconstruction; spatiotemporal

DESCRIPTION (provided by applicant): This project will define the essential mechanics of macropinosome closure. Macropinocytosis and morphologically analogous movements of membrane and the actin cytoskeleton are important in epithelial physiology and host defense against many infections. The successful completion of macropinocytosis always requires closure: the constriction of a region of the cell surface that transforms a cup-shaped invagination of plasma membrane into a discrete membrane-bounded organelle inside the cell. Despite its ubiquity and importance in health and disease, the mechanics and regulation of closure remain largely unexplained. Morphology predicts two distinct stages of macropinosome closure: constriction at the distal margin of cup-shaped, cell surface ruffles, which requires phosphoinositide 3'- kinase (PI3K), followed by a scission that separates the macropinosome from the plasma membrane. The macropinocytic cup can be considered as a transient, self-organized contractile structure at the cell surface. We hypothesize that PI3K organizes actin-myosin-based contractile activities localized to the inner membrane of the macropinocytic cup, which are maintained by a barrier at the distal rim that restricts lateral diffusion of 3' phosphoinositides out of the cup. This hypothesis will be tested by identifying precisely the point of macropinosome closure in macrophages and by localizing barriers to diffusion at various stages of macropinosome formation. Specific Aim 1 will identify the timing and morphology of closure. The point of scission will be identified by measuring access to macropinosomes of externally added, membrane-impermeant fluorescent probes. Instruments and methods developed in this lab will be used to acquire time-resolved, 3-dimensional (4D) reconstructions of constriction and scission in cells expressing fluorescent probes. Specific Aim 2 will identify barriers to lateral diffusion in macropinocytic cups. Morphological studies suggest that during closure the rim of the cup prevents diffusion of inner-leaflet phospholipids out of the cup. A barrier to diffusion at the cup rim could facilitate the local accumulation of 3' phosphoinositides and constrain contractile activities to the inner membrane of the cup. To identify such barriers, fluorescent chimeras of photoactivatable GFP (paGFP) with membrane-anchoring protein domains (paGFP-MEM) will be locally photoactivated in or near macropinosomes or cups. If diffusion of paGFP-MEM in the plane of the bilayer is restricted by cup rims, then fluorescent paGFP-MEM generated inside cups should be constrained to the cup domain of the plasma membrane. Concentration gradients of phosphoinositides within cups and across the cup rim will be measured by quantitative microscopy of cells expressing fluorescent, phospholipid-binding domains. Thus, by defining the spatiotemporal organization of cytoplasm during closure, these studies will create a framework for analyzing molecular mechanisms of closure in macropinosomes and phagosomes. PUBLIC HEALTH RELEVANCE: Pinocytosis is a microscopic drinking activity, present in nearly all living cells, which is especially important in immunity and in kidney function. This work will analyze an unexplained but essential last step in the process, in which an invagination of the cell's plasma membrane closes to form a fluid-filled vesicle inside the cell. The results should have implications for how cells coordinate large-scale activities in their cytoplasm.

描述（由申请人提供）：该项目将定义大型蛋白体闭合的基本机制。膜和肌动蛋白细胞骨架的大型细胞增多症和形态类似运动在上皮生理学和宿主防御许多感染中很重要。大型细胞增多症的成功完成始终需要闭合：细胞表面区域的收缩，该区域将质膜的杯形内陷转化为细胞内部膜结合的细胞器。尽管在健康和疾病中无处不在和重要性，但封闭的力学和调节仍然无法解释。形态可以预测大蛋白体闭合的两个不同阶段：杯形细胞表面荷叶边的远端边缘收缩，这需要磷酸肌醇3'-激酶（PI3K），然后进行训练，然后将大斑肌体与质膜分开。大型细胞杯可以被视为在细胞表面的瞬态，自组织的收缩结构。我们假设PI3K组织了基于肌动蛋白 - 肌球蛋白的收缩活动，该活动位于大型细胞杯内膜上，该活动由远端边缘的屏障维持，限制了杯中3'磷酸磷酸化的横向扩散。该假设将通过准确鉴定巨噬细胞中的大蛋白体闭合点以及通过在大型形成各个阶段扩散的障碍来检验。特定目标1将确定闭合的时机和形态。通过测量对外部添加的，膜覆盖的荧光探针的大分子大体的访问，将确定分裂点。本实验室中开发的仪器和方法将用于获取表达荧光探针的细胞中收缩和SCISSION的时间分辨的3维（4D）重建。特定的目标2将确定在大型细胞杯中横向扩散的障碍。形态学研究表明，在闭合期间，杯子的边缘可以防止内叶磷脂从杯中扩散。在杯边缘扩散的障碍可以促进3'磷酸肌醇的局部积累，并将收缩活性限制为杯子的内膜。为了识别此类障碍，将在巨大症或杯子中或附近局部将具有膜粘蛋白蛋白结构域（PAGFP-MEM）光活化GFP（PAGFP）的荧光嵌合体（PAGFP-MEM）进行。如果PAGFP-MEM在双层平面中的扩散受杯轮辋的限制，则在杯中产生的荧光PAGFP-MEM应限制为质膜的杯子域。通过表达荧光，磷脂结合域的细胞的定量显微镜测量杯中和杯边缘的磷酸肌醇的浓度梯度。因此，通过在闭合过程中定义细胞质的时空组织，这些研究将创建一个框架，用于分析大型粘膜和吞噬体中闭合的分子机制。 公共卫生相关性：皮细胞增多症是一种微观饮酒活性，几乎所有活细胞都存在，这在免疫和肾功能中尤其重要。这项工作将分析该过程中无法解释的但必不可少的最后一步，在该过程中，细胞的质膜的内陷关闭以形成细胞内部的充满液体的囊泡。结果应对细胞如何在其细胞质中坐标大规模活性有影响。